The only mechanism of cAMP action in mammalian cells so far identified is the activation of a protein kinase which catalyzes the phosphorylation of several proteins. A major objective of the proposed program is to establish whether a protein inhibitor of this enzyme that has been identified from in vitro studies may serve as a key physiological regulator of the cAMP system. To evaluate this possibility a program is proposed which will integrate a study of the physicochemical characteristics of the inhibitor protein and its interaction with the components of the protein kinase system with an investigation of parameters which may modify its effective activity in tissues. The direction of physiological modulation by hormones whose actions are mediated by cAMP is quite frequently antagonistic to the actions of insulin and other anabolic hormones. The mechanism of action of insulin remains unknown but the potential that the inhibitor protein may in some way be a component of the insulin regulatory system merits investigation. The specific aims of the proposal include: 1) The purification of the inhibitor protein to homogeneity, and a characterization of its physiochemical properties. 2) A delineation of the parameters that may modulate the amount and/or activity of the inhibitor protein in tissues. 3) An evaluation of whether a correlation exists between the amount of inhibitor protein in cardiac muscle and the degree or rate of phosphorylation of phosphoproteins such as phosphorylase kinase, glycogen synthetase, troponin, and membrane proteins of the sarcoplasmic reticulum or sarcolemma.